Novel therapeutics are needed to treat infections caused by antibiotic-resistant Gram-negative ESKAPE pathogens. DNA Ligase LigA is a validated target for antibacterial discovery, however the translation of LigA competitive inhibitor has been hindered by the rapid emergence of resistance mutations. We will use an innovative substrate-assisted tethered inhibition strategy to develop potent inhibitors of LigA that form a covalent adduct with enzyme-intermediate(s) on the reaction pathway. These inhibitors will thus be uncompetitive with NAD+, circumventing a major mechanism of resistance. We will also seek to identify inhibitors that have long residence times on the enzyme. This will result in increased target suppression at low drug concentration thereby reducing the emergence of resistance while simultaneously improving the therapeutic window of the compounds. In the R21 phase of the proposal, we will synthesize a focused library of oxaboroles (~100-150) guided by our initial modeling and determine the ability of these compounds to inhibit the LigA from K. pneumonia, A. baumannii P. aeruginosa, and E. coli. We will determine MICs against wild-type and efflux-pump mutants of each pathogen (Aim 1). We will develop structure-activity relationships for LigA inhibition using enzyme kinetics, X-ray crystallography and computational approaches (Aim 2) to inform additional compound synthesis. The objective of this aim will be to identify non/uncompetitive inhibitors with IC50 < 1 M Kd < 0.1 M (for E-AMP binding) and MIC < 8 g/ml against efflux-pump strains. In the R33 phase we will focus our efforts on pathogen(s) that demonstrated the greatest sensitivity to the LigA inhibitors in the R21 phase and synthesize additional compounds with improved antibacterial activity against wild-type strain(s). The objective of the R33 phase is to deliver compound(s) with in vivo efficacy (= 1 log CFU decrease) and suitable properties for further optimization and preclinical development. Activities will include improving microbiological activity against wild-type strains, assessment of resistance frequency and in vitro and in vivo DMPK.